Jordan T. Detamore Week 9

Purpose

Defining HIV Structure Research Project

What is your question?
- Will subjects demonstrating a low dS/dN ratio value show high variation and dissimilar relationships between each other when examining their respective amino acid sequences and will subjects demonstrating high dS/dN ratio values show low variation and closer relationships when examining their amino acid sequences both individually and comparatively.
What is your prediction before performing analysis?
- If we extract and compare subjects from the Markham paper who exhibited very high and very low non-synonymous mutation rates and compare the amino acid sequences from their last visits, we will find high variation between them both structurally and functionally because the subjects with lower ratios will have experienced more mutations which caused changes in their amino acid sequencing.
Which subjects, visits, and clones will you use to answer your question?
- Subjects were selected with the same criteria from week 6 presentation Media:HIV1Presentaionzachandshivum.pdf due to selection for "extreme" dS/dN values. Eight subjects were chosen, the four exhibiting the highest dS/dN ratios and the four exhibiting the lowest dS/dN ratios. Due to inconsistencies in subject's visits, we decided to use three clones from the subjects' first (of which everyone's is the same) visit and last (of which some subjects were different) visit. For example, Subject 2's last visit was after 4 years, but subjects 4's last visit was only after 1.5 years. Random selection was used to determine which 3 clone sequences were compared within each visit. The specific clones chosen are:
  - Subject 2
    - First visit clones: 1,6,4
    - Last visit clones: 8,6,1
  - Subject 4
    - First visit clones: 1,2,3
    - Last visit clones: 8,10,7
  - Subject 5
    - First visit clones: 2,5,1
    - Last visit clones: 3,4,5
  - Subject 7
    - First visit clones: 7,9,3
    - Last visit clones: 7,4,5
  - Subject 9
    - First visit clones: 4,2,5
    - Last visit clones: 1,4,7
  - Subject 11
    - First visit clones: 7,5,4
    - Last visit clones: 7,6,3
  - Subject 13
    - First visit clones: 2,1,3
    - Last visit clones: 2,5,6
  - Subject 14
    - First visit clones: 6,1,2
    - Last visit clones: 4,9,13

HIV Structure In-Class Activity

Convert one of your DNA sequences into protein sequences using either the NCBI Open Reading Frame Finder or the ExPASY Translate tool.
- How do you know which of the six frames is the correct reading frame (without looking up the answer)?
  - It is the only sequence that does not have a stop codon. No stop codon should be present because this is the middle segment of a protein sequence.
Find out what is already known about the HIV gp120 envelope protein in the UniProt Knowledgebase (UniProt KB).
- If you search on the keywords "HIV" and "gp120", in the main UniProt search field, how many results do you get?
  - HIV: 723,401 results
  - gp120: 207,983 results
  - HIV gp120: 206,278 results
- Use the entry with accession number "P04578" which corresponds to the reference entry for HIV gp120.
- What types of information are provided about this protein in this database entry?
  - The entry provides information about the functions of both gp120 and gp41 as the HIV virus attacks cells.
We are going to use the PredictProtein server to analyze just the V3 region from Markham et al. (1998).
- Paste one of the amino acid sequences from Markham et al. (1998) into the input field and submit.
- Explore the types of information provided. How does this information relate to what is stored in the UniProt database?
Download the structure file for the paper we read in journal club from the NCBI Structure Database.
- Kwong et al. (1998) structure 1GC1.
- You may also be interested in these other structures from related gp120 structure articles:
These files can be opened with the Cn3D software site that is installed on the computers in the lab (this software is free, so you can download it and use it at home, too.) Alternately, you may choose to use the Star Biochem program to do this portion of your work. Answer the following:
- Find the N-terminus and C-terminus of each polypeptide tertiary structure.
- Locate all the secondary structure elements. Do these match the predictions made by the PredictProtein server?
- Locate the V3 region and figure out the location of the Markham et al. (1998) sequences in the structure.